Alcoholic fermentation yeast and method for producing ethanol using same

ABSTRACT

[Problem] To provide an alcoholic fermentation yeast having resistance to limonene, which is a fermentation inhibitor, and a method for producing ethanol using the alcoholic fermentation yeast. [Solution] An alcoholic fermentation yeast  Saccharomyces cerevisiae  (Deposition No. NITE BP-890), which has resistance to limonene and can grow in the presence of limonene at a concentration of 0.1-0.5 wt %, and a method for producing ethanol using the alcoholic fermentation yeast.

TECHNICAL FIELD

The present invention relates to an alcoholic fermentation yeast havingresistance to limonene, which is a fermentation inhibitor, and to amethod for producing ethanol using the alcoholic fermentation yeast.

BACKGROUND ART

Bioethanol, which is an alternative liquid fuel, can be produced byethanol fermentation of a fruit juice residue extract by yeast addedthereto. Such a technique for converting a juice residue extract, orwaste, to bioethanol by fermentation is very useful because biomass notcompeting with food is used as a starting material. In one known ethanolproduction technique using such a juice residue extract, the juiceresidue extract is produced from a citrus fruit and concentrated by, forexample, centrifugation to obtain a citrus syrup. Then pulp is removedfrom the citrus syrup, and the resultant citrus syrup is subjected toethanol fermentation by Saccharomyces yeast or the like (see, forexample, Patent Literature 1).

CITATION LIST Patent Literature

Patent Literature 1: Japanese Patent Application Laid-Open No.2002-153231

SUMMARY OF INVENTION Technical Problem

Citrus fruits, however, contain limonene (chemical formula: C₁₀H₁₆),which is a terpenoid-based oil component, in an amount of 0.2 to 0.5 wt%. It is known that limonene inhibits ethanol fermentation.

Methods for producing ethanol from a citrus-based starting material(juice residue extract) include a first method in which a citrus syrupobtained by concentrating the juice residue extract is used as thestarting material and a second method in which the juice residue extractitself is used as the starting material without producing a citrussyrup.

The first method has already been contemplated (see, for example,Japanese Patent Application Laid-Open No. 2002-153231). This method hasan advantage in that limonene can be removed in the concentrating step.However, an enormous amount of energy is required in the concentratingstep, and the process of fermentation cannot deal with ahigh-concentration sugar solution, so that the sugar solution must bediluted with water to a sugar concentration of 20% or lower. Therefore,the first method has a problem from the viewpoint of establishing aneconomical ethanol production method.

In the second method, the juice residue extract contains sugar in anamount of about 10%, and this is an optimal concentration forfermentation. Therefore, if the juice residue extract can be fermentedwithout dilution or concentration, an economical ethanol productionprocess can foe established. However, a technique for dealing withlimonene which is a fermentation inhibitor must be established.

The most part of limonene is contained in the peel. However, in a juiceextraction step and an extract extraction step, the fruit including thepeel is mechanically squeezed, and part of limonene is thereby extractedinto the juice or extract. The limonene separated from the peel in theabove steps is mainly in a micelle form in the juice residue extract.According to the results of measurement performed by the inventors, inthe total concentration of limonene contained in a juice residue extractthat is total amount of limonene contained in the peel and limonene in amicelle form (being 0.2 to 0.5 wt %), the concentration of limonene inthe micelle form was very small, being 0.01 to 0.04 wt %.

However, the results of ethanol fermentation experiments performed usinga general yeast with limonene in a micelle (oil) form added to a YPDculture medium showed that the rate of ethanol production decreasedrapidly as the concentration of limonene added increased. For example,when 0.1% v/v of limonene was added, the rate of ethanol production wasgreatly reduced to about 20%. Generally, yeast is present in a sugarsolution as fine solid particles and easily adsorbs oily substances.Therefore, the reason for the above-described experimental results maybe that the limonene (in an oil form) added in a small amount (0.05%v/v) adsorbed to the yeast, causing fermentation inhibition. To removelimonene, which is a fermentation inhibitor, from a juice residueextract, limonene in a solid form, which accounts for 75% or larger ofthe total amount of limonene, must be removed, and limonene in a micelleform present as a light liquid (an oily substance) must also be removed.Even when limonene was removed in advance from a starting material by,for example, three-phase centrifugation treatment, fermentation was notstabilized because the conventional general yeast exhibited an inductionperiod, and the fermentation rate was also low. Therefore,fermentability tended to be unstable due to the influence ofcontamination etc., and it was difficult to maintain high fermentabilityover a long period of time.

The present invention has been made in view of the foregoingcircumstances, and it is an object to provide an alcoholic fermentationyeast having resistance to limonene, which is a fermentation inhibitor,and to provide a method for producing ethanol using the alcoholicfermentation yeast.

Solution to Problem

An alcoholic fermentation yeast according to a first invention thatachieves the above-described object is Saccharomyces having resistanceto limonene.

The alcoholic fermentation yeast according to the first inventionfurther has high-temperature resistance.

The alcoholic fermentation yeast according to the first invention may beSaccharomyces Cerevisiae.

The alcoholic fermentation yeast according to the first invention may beSaccharomyces Cerevisiae (Deposition No. NITE BP-890).

An ethanol production method according to a second invention thatachieves the above-described object uses the alcoholic fermentationyeast according to the first invention.

In the ethanol production method according to the second invention, acitrus may be used as a starting material.

In the ethanol production method according to the second invention, aheavy liquid obtained by subjecting a citrus juice residue extract tothree-phase centrifugation may be used as a starting material.

In the ethanol production method according to the second invention, acitrus juice residue extract containing an acidic material added theretomay be used as a starting material.

In the ethanol production method according to the second invention, theacidic material is preferably nitric acid.

Advantageous Effects of Invention

In the alcoholic fermentation yeast according to any of claims 1 to 4,its resistance to limonene is higher than that of a general yeast, sothat ethanol can be produced efficiently.

In particular, in the alcoholic fermentation yeast according to claim 2,its resistance to high temperature is higher than that of the generalyeast, so that ethanol can be produced efficiently at high temperature.

With the ethanol production method according to any of claims 5 to 9,ethanol can be produced from the starting material containing limonenemore efficiently than when the general yeast is used.

In particular, with the ethanol production method according to claim 6in which the citrus containing limonene is used as the startingmaterial, ethanol can be produced more efficiently than when the generalyeast is used.

In particular, with the ethanol production method according to claim 7,since the concentration of limonene is low, ethanol can be produced moreefficiently.

In particular, with the ethanol production method according to claim 8,since the propagation of unwanted bacteria is suppressed, ethanol can beproduced more efficiently.

In particular, with the ethanol production method according to claim 9,there is no fear of corrosion of steel materials used in an ethanolproduction apparatus.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram illustrating the base sequence of a BP-890 strainaccording to Example 1 of the present invention.

FIG. 2 is a microscope photograph of the BP-890 strain (1000×magnification).

FIG. 3 is a graph showing the limonene resistance of the BP-890 strain.

FIG. 4 is a graph showing the high-temperature resistance of the BP-8 90strain.

FIGS. 5(A) and 5(B) are a graph showing the batch fermentationcharacteristics of the BP-890 strain and a graph showing the batchfermentation characteristics of a general yeast, respectively.

FIG. 6 is a graph showing the ethanol fermentation characteristics ofthe BP-890 strain in fed batch culture.

FIG. 7 is a process chart showing steps in an ethanol production methodin Example 3 of the present invention.

FIG. 8 is a diagram illustrating the schematic configuration of acontinuous fermentation apparatus used in an ethanol fermentation step.

FIG. 9 is a graph (1) showing the results of ethanol production.

FIG. 10 is a graph (2) showing the results of ethanol production.

FIGS. 11(A) and 11(B) are a graph (3) showing the results of ethanolproduction by the general yeast and a graph (4) showing the results ofethanol production by the BP-890 strain, respectively.

DESCRIPTION OF EMBODIMENTS

Embodiments of the present invention will next be described withreference to the accompanying drawings, in order to provide anunderstanding of the present invention. An alcoholic fermentation yeastaccording to an embodiment of the present invention is Saccharomyceshaving limonene resistance. This alcoholic fermentation yeast furtherhas high-temperature resistance. When ethanol is produced using thealcoholic fermentation yeast according to this embodiment, tangerineorange (an example of citrus fruits) is used as a starting material.However, the technical scope of the present invention is not construedto be limited by the following embodiments.

Hereinafter, the “limonene resistance” means a property of a yeast thatallows ethanol fermentation at a yield of 86% or higher in the presenceof limonene at a concentration of 0.1to 0.5 wt % in a starting material.The “high-temperature resistance” means a property of a yeast thatallows ethanol fermentation at a yield of 86% or higher in anenvironment with a fermentation temperature of 40° C. or higher.Saccharomyces Cerevisiae with no limonene resistance and nohigh-temperature resistance is referred also to as a “general yeast.”

The present invention will next be described by way of Examples.

EXAMPLE 1 Isolation of Novel Yeast

A tangerine orange juice residue extract was collected from a productionline in a factory for juice of citrus fruits harvested in Ehimeprefecture. This tangerine orange juice residue extract was diluted by afactor of 1,000, and the dilution was spread over a YPD agar culturemedium (containing 1% of yeast extract, 2% of peptone, and 2% ofglucose) and cultured at: 30° C. for about 2 days.

Various yeasts forming colonies on the culture petri dish were pickedand subjected to pure culture on YPD culture mediums (containing 2% ofyeast extract, 2% of peptone, and 10% of glucose, pH: 6.5 to 7.0).

Screening of Yeasts

A yeast having limonene resistance was screened from the pure culturedyeasts.

More specifically, ethanol fermentation was performed using severalsugar solutions as starting materials with different limoneneconcentrations within the range of 0.02 to 0.2% v/v. Then strains havinghigh ethanol fermentability in the sugar solutions with differentlimonene concentrations were selected as limonene resistant strains.Among these limonene resistant strains, a strain having particularlyhigh limonene resistance was deposited (Deposition No. NITE BP-890). Inthe present description, the deposited strain is hereinafter referred toas a “BP-390 strain.” The BP-890 strain has been deposited in thefollowing depositary by the application filed by the present applicants.

(1) Name: NITE Patent Microorganisms Depositary (NPMD)

(2) Address; 2-5-8 Kazusakamatari, Kisarasu-shi, Chiba 292-0818 Japan

(3) Phone number: 0438-20-5580

(4) Deposition No.: NITE BP-890

(5) Deposition No. at the time of original deposit: NITE P-890

(6) Date of original deposit: Jan. 28, 2010

(7) Conversion date under the Budapest Treaty: Jun. 3, 2011

Identification of BP-890 Strain

DNA was extracted from the BP-890 strain, and its D2LSUrDNA region wasamplified by PCR. The base sequence of the obtained PCR product wasdetermined by direct sequencing (see FIG. 1). On the basis of this basesequence, a BLAST search was performed on the D2LSUrDNA sequences ofknown microorganisms in a database to identify microorganisms estimatedas closely-related species. As shown in the results in TABLE 1, theBP-890 strain was found to be a Saccharomyces Cerevisiae yeast.

TABLE 1 BLAST RESULT FOR BP-890 STRAIN Query Max ident AccessionDescription Max score Total score coverage (HOMOLOGY) AB362220.1Saccharomyces cerevisiae gene for 26S ribosomal RNA, 578 578 99% 99%partial sequence, strain: TY-2 DQ888227.1 Synthetic construct clonepNOY373 35S ribosomal RNA, 578 578 99% 99% 18S ribosomal RNA, 5.8Sribosomal RNA, 25S ribosomal RNA, and 5S ribosomal RNA, completesequence DQ674258.1 Saccharomyces cerevisiae strain L68 28S 578 578 99%99% ribosomal RNA gene, partial sequence AY497669.1 Saccharomycescerevisiae strain CBS 1171 26S 578 578 99% 99% ribosomal RNA gene,partial sequence J01355.1 Saccharomyces cerevisiae 25S 578 578 99% 99%ribosomal RNA gene, complete sequence AY046154.1 Saccharomycescerevisiae NRRL Y-12632 578 578 99% 99% 26S ribosomal RNA gene, partialsequence AY529515.1 Saccharomyces cerevisiae isolate 40 26S 578 578 99%99% ribosomal RNA gene, partial sequence Z73326.1 S.cerevisiaechromosome XII reading frame ORF YLR154c 578 578 99% 99% U53879.1Saccharomyces cerevisiae chromosome XII cosmid 9634 578 1156 99% 99%GQ121701.1 Saccharomyces cerevisiae strain IMAU6Y152(DX11-2) 576 576 98%100% 26S large subunit ribosomal RNA gene, partial sequence GQ121699.1Saccharomyces cerevisiae strain IMAU6Y150(DX10-3) 576 576 98% 100% 26Slarge subunit ribosomal RNA gene, partial sequence GQ121698.1Saccharomyces cerevisiae strain IMAU6Y149(DX10-2) 576 576 98% 100% 26Slarge subunit ribosomal RNA gene, partial sequence GQ121692.1Saccharomyces cerevisiae strain IMAU6Y143(DX8-1) 576 576 98% 100% 26Slarge subunit ribosomal RNA gene, partial sequence GQ121690.1Saccharomyces cerevisiae strain IMAU6Y141(DX7-3) 576 576 98% 100% 26Slarge subunit ribosomal RNA gene, partial sequence GQ121688.1Saccharomyces cerevisiae strain IMAU6Y139(DX7-1) 576 576 98% 100% 26Slarge subunit ribosomal RNA gene, partial sequence

Mycological Properties

The BP-8 90 strain has the following mycological properties.

1. Cell shape: Spherical (see FIG. 2)

2. Colony shape: White (no gloss), wrinkled.

3. Proliferation form: multilateral budding

4. Optimal growth temperature; 30° C.

5. Optimal growth pH: 4.0

6. Cohesiveness: No

7. Fermentable sugars: sucrose, glucose, and fructose

EXAMPLE 2 Limonene Resistance Test

The BP-890 strain was subjected to batch fermentation (shake culture)with limonene added at different concentrations to examine ethanolfermentability. The experimental conditions are as follows.

Experimental Conditions

1. Fermentation temperature: 30° C.

2. Shake rate: 120 rpm (amplitude: 10 mm)

3. Fermentation time: 24 hours

4. Starting material: YPD culture medium

The results are shown in FIG. 3. In. FIG. 3, the horizontal axisrepresents the amount (% v/v) of limonene added to the YPD culturemediums, and the vertical axis represents the rate of ethanol production(%).

As is clear from the figure, with the general yeast, as theconcentration of limonene increased, the ethanol production ratedecreased. However, with the BP-890 strain, until the concentration oflimonene added was about 0.1% v/v, the ethanol production rate washigher than 90%, and the ethanol production rate did not decrease.

High-Temperature Resistance Test

24 Hour batch fermentation experiments were performed using YPD culturemediums at fermentation temperatures of 30° C., 37° C., 40° C., 42° C.,and 45° C. The experimental conditions are as follows.

Experimental Conditions

1. Shake rate; 120 rpm (amplitude: 10 mm)

2. Fermentation time: 24 hours

3. Starting material: YPD culture medium

The results are shown in FIG. 4. In the figure, the horizontal axisrepresents temperature, and the vertical axis represents the rate ofethanol production. There are portions in which the ethanol productionrate is slightly higher than 100%. These large ethanol production ratesmay be due to measurement error.

As is clear from the figure, the general yeast was capable offermentation only up to a fermentation temperature of 37° C. However,the BP-890 strain was capable of fermentation up to a fermentationtemperature of 40° C. at a high yield of 90% or higher. Morespecifically, the BP-890 strain was found to have high-temperatureresistance. In addition, the ethanol production rate of the BP-890strain was about 70% even at 42° C., and the BP-890 was found to becapable of fermentation even at 42° C. at a relatively high yield.

Ethanol Fermentation Test [1]

Batch culture was performed in the presence of limonene to examineethanol production and growth of the yeasts. The experimental conditionsare as follows.

Experimental Conditions

1. Fermentation temperature: 30° C.

2. Stirring rate: 120 rpm

3. Fermentation time: 24 hours

4. Starting material: Heavy liquid obtained by three-phasecentrifugation of tangerine orange (Citrus unshiu) juice residue extract(pH is adjusted to 3.5)

The results for the BP-890 strain are shown in FIG. 5(A). In FIG. 5, thehorizontal axis represents time, and the vertical axis represents theconcentrations (g/L) of sucrose, glucose, fructose, and ethanol and theconcentration (cells/ml) of the yeast.

As shown in FIG. 5(B), the general yeast exhibited an induction periodof about 8 hours. However, the BP-890 strain exhibited no inductionperiod, and the proliferation started immediately after the start ofculture. The yeast proliferation rate and the ethanol production ratewere higher with the BP-890 strain than with the general yeast. Becauseof these characteristics, the use of the BP-890 strain for ethanolfermentation allows culture to be performed more stably and in a shorterperiod of time than when the general yeast is used and also allowsefficient fermentation with a short residence time to be performed.

Ethanol production was similarly examined using the following threestarting materials prepared from this tangerine juice residue extractunder different conditions, i.e., with or without three-phasecentrifugation and with or without addition of nitric acid.

(1) Tangerine orange juice residue extract having a pH of 3.5 adjustedby addition of nitric acid (no three-phase centrifugation)

(2) Heavy liquid obtained by three-phase centrifugation of tangerineorange juice residue extract (no pH adjustment)

(3) Tangerine orange juice residue extract (no three-phasecentrifugation and no pH adjustment)

The effect of the three-phase centrifugation is removal of limonene. TheBP-890 strain has limonene resistance. Therefore, it seems that theremoval of limonene is unnecessary. However, the condition that whetheror not a tangerine orange juice residue extract has been subjected tothree-phase centrifugation is considered because of the followingreasons: (1) although the BP-890 strain has limonene resistance, theethanol production rate is expected to be further improved when thelimonene concentration is low, and (2) limonene cannot be removedcompletely by three-phase centrifugation. The effect of the addition ofnitric acid is suppression of propagation of unwanted bacteria. Thedetails of the effects of three-phase centrifugation and the addition ofnitric acid will be described later.

The results under these conditions are shown in TABLES 2 to 4.

TABLE 2 ETHANOL FERMENTATION TEST RESULTS (CENTRIFUGATION: NO, pHADJUSTMENT: YES) TOTAL ETHANOL OF THREE PRODUCTION SUCROSE GLUCOSEFRUCTOSE SUGARS ETHANOL RATE [g/L] [g/L] [g/L] [g/L] [g/L] [%] GENERALBEFORE 22.9 30.4 30.0 83.3 0.0 — YEAST FERMENTATION AFTER 0.0 0.0 1.21.2 39.2 92.1 FERMENTATION BP-890 BEFORE 27.0 20.5 26.3 73.8 4.9 —STRAIN FERMENTATION AFTER 1.9 0.0 2.3 4.1 43.9 103.5 FERMENTATION

TABLE 2 shows the results of the measurement of the concentrations (g/L)of sucrose, glucose, fructose, and ethanol before and after fermentationand the ethanol production rate (%) for each of the general yeast andthe BP-890 strain.

TABLE 3 ETHANOL FERMENTATION TEST RESULTS (CENTRIFUGATION: YES, pHADJUSTMENT: NO) AVERAGE TOTAL ETHANOL ETHANOL OF THREE PRODUC- PRODUC-SUCROSE GLUCOSE FRUCTOSE SUGARS ETHANOL TION RATE TION RATE [g/L] [g/L][g/L] [g/L] [g/L] [%] [%] GENERAL BEFORE 23.2 15.1 14.1 52.5 0.0 — —YEAST FERMENTATION AFTER 0.0 5.3 1.8 7.0 24.3 90.7 89.9 FERMENTATIONAFTER 0.0 6.3 1.3 7.7 23.9 89.0 FERMENTATION BP-890 BEFORE 22.5 14.613.6 50.7 0.0 — — STRAIN FERMENTATION AFTER 0.0 0.0 2.0 2.0 23.6 90.994.1 FERMENTATION AFTER 0.0 0.0 2.1 2.1 25.2 97.3 FERMENTATION

TABLE 3 shows the results of the measurement of the concentrations (g/L)of sucrose, glucose, fructose, and ethanol before and after fermentationand the ethanol production rate (%) for each of the general yeast andthe BP-890 strain.

Data were measured twice after fermentation and represented as “afterfermentation (1)” and “after fermentation (2).” An average ethanolproduction rate (%) is the average of the two measured ethanolproduction rates (%).

TABLE 4 ETHANOL FERMENTATION TEST RESULTS (CENTRIFUGATION: NO, pHADJUSTMENT: NO) AVERAGE TOTAL ETHANOL ETHANOL OF THREE PRODUC- PRODUC-SUCROSE GLUCOSE FRUCTOSE SUGARS ETHANOL TION RATE TION RATE [g/L] [g/L][g/L] [g/L] [g/L] [%] [%] GENERAL BEFORE 25.1 23.9 28.3 77.3 0.0 — —YEAST FERMENTATION AFTER 0.0 11.2 2.2 13.4 15.0 38.1 36.0 FERMENTATIONAFTER 0.0 12.5 6.5 19.0 13.4 34.0 FERMENTATION BP-890 BEFORE 25.1 23.928.3 77.3 0.0 — — STRAIN FERMENTATION AFTER 0.0 9.1 1.7 10.8 21.5 54.450.9 FERMENTATION AFTER 0.0 10.8 3.1 13.9 18.7 47.3 FERMENTATION

TABLE 4 shows data similar to the data in TABLE 3.

As is clear from the results in TABLES 2 to 4, the ethanol productionrates were higher with the BP-890 strain than with the general yeast. Inparticular, for the tangerine orange juice residue extract with nocentrifugation treatment and no pH adjustment (untreated startingmaterial), when the BP-890 strain was used, the ethanol production ratewas higher by 15% than that with the general yeast (see TABLE 4). InTABLE 2, ethanol was present before fermentation. This is becauseethanol contained in a cultured yeast solution added to the startingmaterial was detected.

Ethanol Fermentation Test [2]

Fed batch culture was performed using a heavy liquid obtained bythree-phase centrifugation of a tangerine orange juice residue extractas a starting material to examine ethanol production and the grown ofyeast. The experimental conditions are as follows.

Experimental Conditions

1. Temperature: 30° C.

2. Stirring rate: 150 rpm

3. Starting material supply: 3.3 ml/min

4. Reactor volume: 5 L

5. Amount of air blown: 0.05 vvm

6. Amount of yeast, added: 3.0%v/v

7. Starting material: tangerine orange (Citrus unshiu) juice residueextract

The results are shown in FIG. 6. In FIG. 6, the horizontal axisrepresents time, and the vertical axis represents the ethanolconcentration (g/L) and the yeast concentration (cells/bath).

As is clear from the figure, the solution subjected to three-phasecentrifugation and having a low limonene concentration exhibited aninduction period of about 4 hours when the general yeast was used.However, when the BP-890 strain was used, there was no induction period,and proliferation started immediately after the start of culture. Withthe BP-8 90 strain, ethanol production was started immediately after thestart of culture and reached a peak at 15 hours. More specifically, withthe BP-890 strain, the ethanol production rate was found to be higherthan that with the general yeast. In other words, the BP-890 strainexhibits no induction period even when used for a starting materialsubjected to three-phase centrifugation and having a low limoneneconcentration and allows stable fermentation.

EXAMPLE 3 Ethanol Production [1] Using BP-890 Strain

Ethanol was produced using the BP-890 strain and iyokan as an example ofthe starting material. A description will first be given of a productionmethod.

The ethanol production method includes an initial process and a laterprocess as shown in FIG. 7. The initial process includes a juicesqueezing step P1, a mixing step P2, and an extract extracting step P3.The later process includes a three-phase centrifugation step P4, an acidmixing step P5, a fermentation step P6, and a distillation step P7. Eachof these steps will next be described.

The juice squeezing step P1 is a step of squeezing the iyokan using ajuice squeezing machine such as an in-line type or chopper pulper typemachine. In this step, juice residue 11 and juice 12 are produced.

The mixing step P2 is a step of mixing the juice residue 11 produced inthe juice squeezing step P1 with slaked lime being an example of analkaline material. Since the juice residue 11 produced in the juicesqueezing step P1 is in a gel or slurry form, an extract cannot beextracted efficiently using a press. However, the addition and mixing ofthe slaked lime allows the extract to be extracted efficiently.

The extract extracting step P3 is a step of pressing the juice residue11 that has been treated so as to be easily pressed in the mixing stepP2 to thereby subject the juice residue 11 to dewatering treatment. Inthis step, a juice residue extract 13 and pomace 14 are produced.

The three-phase centrifugation step P4 is a step of subjecting the juiceresidue extract 13 produced in the extract extracting step P3 tothree-phase centrifugation to form a heavy liquid 15, a light liquid 16,and solids 17.

In the three-phase centrifugation step P4, limonene, or a fermentationinhibitor, contained in the juice residue extract 13 is removed togetherwith the light liquid (oily substance) 16 and the solids 17. In thismanner, the limonene concentration in the liquid part of the heavyliquid 15 can be reduced to 0.01 wt % or lower, and the overallconcentration of limonene including limonene in solids in the heavyliquid 15 can be reduced to 0.1 wt % or lower.

By subjecting the juice residue extract 13 to three-phase centrifugationto remove the fermentation inhibitor in this step, the proliferationproperties of the yeast during ethanol fermentation is improved. Thefermentability is thereby stabilized, and the ethanol production rate isincreased.

Since the three-phase centrifuge used in this step separates the lightliquid and heavy liquid with a small difference in specific gravity, itis preferable that the three-phase centrifuge used in this step be adisk-type centrifuge that can have a large sedimentation area. When acentrifugal force of 5,000 G or larger is available, the separation canbe industrially performed.

The acid mixing step P5 is a step of mixing the heavy liquid 15 producedin the three-phase centrifugation step P4 with nitric acid which is anexample of an acidic material to form an acid-mixed solution 18. Morespecifically, in this step, 60% concentrated nitric acid is added to theheavy liquid 15 produced in the three-phase centrifugation step P4, andthe mixture is stirred and mixed using a stirring pump or a stirrer. ThepH of the heavy liquid 15 is thereby adjusted to 3.5, and the acid-mixedsolution 18 is produced.

Since the specific gravity of nitric acid is larger than the specificgravity of the heavy liquid 15 produced in the three-phasecentrifugation step P4, the mixture is continuously or regularly stirredafter the addition of nitric acid.

In this step, the amount of unwanted bacteria in the acid-mixed solution18 is reduced to about 1/10 to 1/1,000 of the amount before the additionof nitric acid. The pH after adjustment is set in consideration of thefacts that (1) the optimal pH for the life of unwanted bacteria is 5.0to 7.0 and (2) the optimal pH for the growth of the yeast necessary forethanol fermentation in the next step is 3.5 to 6.0. More specifically,in this step, the pH is adjusted such that the yeast can grow with thepropagation of unwanted bacteria suppressed. Such a pH is, for example,3.0 to 4.0.

The fermentation step P6 is a step of adding the SaccharomycesCerevisiae BP-890 strain to the heavy liquid 15 (the acid-mixed solution18) with its pH adjusted in the acid mixing step P5 to thereby performethanol fermentation. The fermentation in this step is continuousfermentation but may be batch fermentation.

The distillation step P7 is a step of distilling the ethanolfermentation solution produced in the fermentation step P6. In thisstep, ethanol 19 can be purified.

Ethanol was produced using the BP-890 strain according to theabove-described production steps.

First, in the juice squeezing step P1, iyokan used as the startingmaterial was squeezed. Next, in the mixing step P2, slaked lime wasmixed into the produced juice residue 11. Then, in the extractextracting step P3, the juice residue 11 containing slaked lime mixedthereinto was pressed to produce a juice residue extract 13. Next, inthe three-phase centrifugation step P4, the juice residue extract 13produced in the extract extracting step P3 was subjected to athree-phase centrifuge to separate a heavy liquid 15. Then, in the acidmixing step P5, nitric acid was added to the heavy liquid 15 to adjustits pH to 3.5, and the mixture was stirred for 2 hours to produce anacid-mixed solution 18. Then, in the fermentation step P6, continuousfermentation was performed.

A continuous fermentation apparatus 20 used in the fermentation step P6will next be described. As shown in FIG. 8, in the fermentation step P6,the acid-mixed solution 18 (pH: 3.5) used as the starting material wasfed from a pump 21 to a bioreactor 22. In the bioreactor 22, continuousfermentation (average residence time: 24 hours) was performed. Thefermented solution overflowed from the upper part of the continuousfermentation apparatus 20. During fermentation, sterilized air was blowninto the fermented solution in the bioreactor 22. The fermented solutionwas continuously stirred using a three-blade impellor 25. Thetemperature inside the bioreactor 22 was maintained by a thermostaticbath 26.

The detailed fermentation conditions are as follows.

Continuous Fermentation Conditions

1. Fermentation temperature: 30° C.

2. Stirring rate: 150 rpm

3. Fermentation time: 24 hours (residence time)

4. Amount of yeast added: 3.0% v/v

5. Reactor volume: 5 L

6. Dilution ratio: 0.04 (l/h)

7. Amount of sterilized air blown: 0.05 vvm

8. Other: the dilution ratio was changed in the range of 0.014 to 0.04during continuous fermentation

The results are shown in FIG. 9. In FIG. 9, the horizontal axisrepresents time elapsed, and the vertical axis represents theconcentrations (g/L) of sucrose, glucose, fructose, and ethanol, thetotal concentration (g/L) of all the sugars, and the concentration(cells/ml) of the yeast.

In this Example, the dilution ratio D was changed in the range of 0.014to 0.04. However, ethanol was produced stably irrespective of thechanges in the dilution ratio D. Generally, when the dilution ratio D ischanged during continuous fermentation using the general yeast,fermentability becomes unstable, and the amount of ethanol produced andthe amount of the yeast decrease as the time elapses, so thatfermentation failure occurs. The average ethanol production rate was105.4%. The average ethanol production rate exceeded 100%. This may bebecause of the following two reasons. First, the sugars taken intoaccount to compute the average ethanol production rate were sucrose,glucose, and fructose, but the yeast fermented sugars other than thesesugars. Second, the average ethanol production rate was computed to belarger due to measurement error.

EXAMPLE 4 Ethanol Production [2] Using BP-890 Strain

This Example is different from Example 3 in the following two points.The centrifugation step P4 was omitted, and the range of the changes inthe dilution ratio D was changed. More specifically, after a juiceresidue extract was produced in the extract extracting step P3, thecentrifugation step P4 was omitted, and nitric acid was added to thejuice residue extract in the acid mixing step P5 to produce anacid-mixed solution having a pH of 3.5. The acid-mixed solution was fedto the bioreactor 22 to perform the fermentation step P6. Theconcentration of limonene in the acid-mixed solution prepared byadjusting the pH of the juice residue extract 13 (the total of theconcentration of limonene adhering to the solids and the concentrationof limonene present in the solution) was 0.2% v/v.

The results are shown in FIG. 10. In FIG. 10, the horizontal axisrepresents time elapsed (h), and the vertical axis represents theconcentrations (g/L) of sucrose, glucose, fructose, and ethanol, thetotal concentration (g/L) of all the sugars, and the concentration(cells/ml) of the yeast.

In this example, the dilution ratio D was changed in the range of 0.04to 0.08. However, ethanol was produced stably irrespective of thechanges in the dilution ratio D. The average ethanol production rate was88.5%.

EXAMPLE 5 Ethanol Production [3] Using BP-890 Strain

In this example, the operating conditions of the continuous fermentationapparatus 20 (see FIG. 8) are different from those in Example 3 above.More specifically, the general yeast and the BP-890 strain were used,and continuous fermentation was performed for each of these yeasts underthe operating conditions in which the fermentation treatment wassuspended and resumed repeatedly according to the operation of the juicefactory. The concentration of limonene in the acid-mixed solutionprepared by adjusting the pH of the juice residue extract 13 was about0.03 vol %.

The details of the operating conditions of the continuous fermentationapparatus 20 are as follows.

Operating Conditions

1. A fermentation period (12 hours) in which ethanol fermentation isperformed and a suspended period (12 hours) in which the supply of thestarting material is stopped to suspend the fermentation are repeated(the processing of the starting material is performed for a half day,and the processing is suspended for another half day).

2. The dilution ratio D during the fermentation period is 0.03 (averageresidence time: 12 hours).

3. During the suspended period, the temperature inside the bioreactor 22is maintained at 30° C. The stirring and the blowing of sterilized airare continued (only the supply of the starting material is suspended).

The results using the general yeast and the BP-890 strain are shown inFIGS. 11(A) and 11(B). In FIG. 11(A), the horizontal axis representstime elapsed (h), and the vertical axis represents the concentrations(g/L) of sucrose, glucose, fructose, and ethanol, the totalconcentration (g/L) of ail the sugars, the total concentration (g/L) ofall the sugars in an inflow solution, and the concentration (cells/ml)of the yeast.

In FIG. 11(B), the horizontal axis represents time elapsed (h), and thevertical axis represents the concentrations (g/L) of sucrose, glucose,fructose, and ethanol, the total concentration (g/L) of all the sugars,the total concentration (g/L) of these three sugars in an inflowsolution, and the concentration (cells/ml) of the yeast.

In this Example, when the supply of the starting material was suspendedin the presence of limonene which is the growth inhibitor, the growth ofthe general yeast with no limonene resistance was inhibited by limoneneand also adversely affected by malnutrition etc. This caused asignificant reduction in the growth rate of the yeast and the death ofthe yeast, and the amount of the yeast thereby decreased. Then thefermentability deteriorated. However, with the BP-890 strain, noreduction in the amount of the yeast was found even when the supply ofthe starting material was suspended, and high fermentability wasmaintained stably. More specifically, stable operation is impossiblewith the general yeast under the conditions in which the supply of thestarting material is suspended temporarily, for example, suspended atnight according to the operation of a juice factory. However, the BP-890strain having limonene resistance was found to allow stable fermentationeven under such conditions.

The present invention is not limited to the above-described embodimentsand Examples, and any modification can be made so long as the gist ofthe invention is not changed.

In Examples 3 and 4 described above, ethanol fermentation was performedusing the juice residue extract 13 obtained in the extract extractingstep P3. However, ethanol fermentation (solid fermentation) may beperformed using, as the starting material, the juice residue 11 obtainedin the juice squeezing step P1.

REFERENCE SIGNS LIST

11: juice residue, 12: juice, 13: juice residue extract, 14: pomace, 15:heavy liquid, 16: light liquid, 17: solids, 18: acid-mixed solution, 19:ethanol, 20: continuous fermentation apparatus, 21: pump, 22:bioreactor, 25: three-blade impellor, 26: thermostatic bath

1-9. (canceled)
 10. An ethanol production method comprising: squeezingcitrus to produce a juice residue; mixing the juice residue with slakedlime; dewatering the juice residue mixed with the slaked lime to producea juice residue extract; mixing the juice residue extract with nitricacid to form an acid-mixed solution with a pH of 3.0 to 4.0 withoutsubjecting the juice residue extract to a three-phase centrifugation toform a heavy liquid, a light liquid, and solids; and adding an alcoholicfermentation yeast having a limonene resistance to the acid-mixedsolution to perform alcoholic fermentation.
 11. The method according toclaim 10, wherein the alcoholic fermentation is continuous fermentationwhich is performed at a dilution ratio of from 0.04 to 0.08.
 12. Themethod according to claim 11, wherein the alcoholic fermentation yeastis Saccharomyces Cerevisiae (Deposition No. NITE BP-890).
 13. The methodaccording to claim 11, further comprising subjecting the juice residueextract to the three-phase centrifugation to form the heavy liquid, thelight liquid, and the solids by using a centrifuge after dewatering thejuice residue and before mixing with the nitric acid, wherein the nitricacid is mixed into the heavy liquid instead of the juice residueextract.
 14. The method according to claim 13, wherein a concentrationof limonene in the heavy liquid is 0.01 wt % or lower in the three-phasecentrifugation.
 15. The method according to claim 14, wherein thecentrifuge is a disk-type centrifuge.
 16. The method according to claim15, wherein a centrifugal force of the disk-type centrifuge is 5000 G orlarger.